Project Summary The strongest established risk factor for the progressively fatal disease pulmonary arterial hypertension (PAH) is female sex (~3:1 ratio of females: males). Elevated circulating estrogen levels, and enhanced estrogen signaling, are a feature of PAH. Our evidence suggests that exuberant estrogen signaling causes a perturbation of mitochondrial function and energy substrate utilization in both sexes. However, not all PAH patients have elevated estrogens, and we don?t know the affinity of the pulmonary vascular bed for estrogens. In preliminary studies of PAH patients, estradiol (E2) levels were higher pre- than post-pulmonary capillary bed, suggestive of E2 uptake by the lungs. While this transpulmonary (TP) gradient was variable among patients, those with a more negative TP gradient had more severe hemodynamic metrics at diagnosis. E2 and other estrogens, such as 16?-hydroxyestrone (16?OHE1), signal via the canonical estrogen receptors (ESR? and ESR?). In a transgenic mouse model of PAH, we found that administration of 16?OHE1 significantly increased PAH penetrance concomitant with features of oxidant stress, insulin resistance and mitochondrial dysfunction?all characteristics we and others have described in humans. ESR signaling also reduced PPAR? expression via a reduction in PGC1?. With tamoxifen, a direct ESR antagonist, we prevented the cellular metabolic defects and pulmonary vascular phenotype in our transgenic murine model system. Tamoxifen is a well-tolerated FDA-approved drug and the most commonly used hormonal therapy to antagonize ESRs. In our model system, we measured the degree of ESR antagonism by tamoxifen using a PET scan with estrogen tracer. In humans, we have applied this approach to those treated with tamoxifen for breast cancer?providing an opportunity to associate the degree of antagonism with therapeutic response. Our central hypothesis is that estrogen antagonism by tamoxifen will be a safe therapeutic approach for PAH, and that characteristics of a highly estrogenic profile in the blood and lungs will identify PAH patients likely to have the most benefit. We will test this hypothesis with these Specific Aims: (1) Test the hypothesis that estrogen antagonism with tamoxifen is safe in humans with PAH, using a 24 week proof-of-concept safety trial comparing tamoxifen (n=12) to placebo (n=12). (2) Determine the phenotype profile of subjects with PAH for whom estrogen antagonism may be an effective therapeutic approach?TP E2 levels and lung ESR density will be determined at diagnosis, and associated with disease severity. (3) Test the hypothesis that estrogen signaling drives mitochondrial fragmentation and oxidative damage leading to pulmonary hypertension via regulation of PGC1?, to uncover novel therapeutic approaches that target only the deleterious effects of estrogens in PAH. Ultimately, we aim to demonstrate the safety of direct ESR antagonism, those patients most likely to benefit, and the cellular mechanisms that drive estrogen signaling to promote PAH. This proposal should provide the foundation for a definitive trial of estrogen antagonism using a precision medicine approach.